The present study was undertaken to develop a rapid, quantitative, in vitro system for evaluating antiviral agents, neutralizing antibodies and various cytokine effects on HIV-1 replication. A system was developed to simultaneously test the effects of various agents on 1) the replication of representative strains of HIV, 2) virus transmitted free of cells or associated with cells, and 3) HIV transmitted to T-cells and monocytes. The test system uses indicator T-cell (H938) and monocyte (U38) cell lines containing stably integrated copies of the HIV-LTR-CAT gene. Increased CAT activity signals increased viral tat protein production after viral infection. For virus inocula, H9 T-cells were similarly infected with one of four representative strains of HIV-1 (IIIB, MN, RF, Z). Infected cells and culture supernatants were cryopreserved for use as cell-associated and cell-free virus inoculum, respectively. CAT signals produced in indicator cells (1.4 x104 per well) were determined by diffusion assay at several times after various concentrations of cell-associated and cell-free virus inocula were added. There was a range of inocula over which CAT enzyme activity was proportional to inoculum added. After only 3 days of culture, cell-associated infection of T-cells produced an average CAT activity 322- fold over controls, while cell-free inoculum increased CAT activity 200- fold over controls. Increased CAT activity 2-3-fold were observed in monocyte cells after 14 days in culture. Several neutralizing antibodies induced by a synthetic peptide to env of the HIV-IIIB were titered against the four strains of HIV (adjusted to produce a 25-fold increase) presented cell-free or associated with cells in the test system. Antibody effects on HIV infection of indicator cells were clearly distinguishable by virus strain, virus presentation and target cell type. I will be participating in the Antibody Serological Project under Div. of AIDS, NIAID. Study reagents will provide an opportunity to compare data with other neutralization assays. Studies are planned to characterize neutralizing antibodies of AIDS patients throughout disease and therapy and antibodies elicited in response to vaccines.